Double-strand DNA breaks (DSBs) occur in all cells multiple times per day. Homologous recombination (HR) and nonhomologous DNA end-joining (NHEJ) are the two primary pathways for repairing DSBs. However, NHEJ is the more general pathway because it does not require a homologous donor, and NHEJ is sufficiently flexible that it can join almost any pair of incompatible DNA ends. The flexibility of NHEJ is essential because natural causes of DSBs (ionizing radiation, free radicals, enzyme malfunction) generate DNA ends with diverse chemical and structural configurations. Hence, that flexibility is well-suited for the task, but the price that we pay for that flexibility is that NHEJ causes DNA sequence changes at most sites where it functions (in humans and other vertebrates). Hence, NHEJ generates somatic cell mutations that cause cancer and likely contribute to aging. The flexibility of NHEJ represents one of the most sophisticated protein:DNA interaction pathways. Though we know most of the proteins that participate in NHEJ and know, in broad terms, what they do, we do not have a clear picture of how they function together, their spatial configuration, or the temporal relationships. With a clearer mechanistic and structural picture of human NHEJ, we will be in a position to develop small molecule inhibitors that may be useful for treating cancer (as chemotherapy or as a radiation sensitizer). Aims 1 and 2 of this proposal focus precisely on the issues of structural and spatial relationships among the NHEJ proteins and with the DNA ends. Aim 3 tests the hypothesis that the NHEJ ligase complex is important for making key end-to-end contacts during NHEJ. Aim 4 describes development of a more robust NHEJ reconstitution. In Aim 4, that system is then used to identify any remaining NHEJ factors by testing for enhancement of activity. Aim 5 describes analysis of how histone octamers influence the nuclease, polymerase, and ligase activities of NHEJ. These studies will markedly improve our understanding of how NHEJ participates in processes such as chromosomal translocations that are of key importance in cancer etiology and how NHEJ might be made 'druggable' for therapeutic purposes.